Occurrence and genetic characterization of Echinococcus granulosus sensu lato from domestic animals in Central Iran

Background The species complex of Echinococcus granulosus sensu lato (s.l.) causes cystic echinococcosis distributed worldwide. There is no genotype information from hydatid cysts in the intermediate hosts in Central Iran. Therefore, in this study, we analyzed the hydatid cysts in livestock slaughtered in an abattoir in this region. Six hundred fifty-seven hydatid cysts were isolated from 97 animals, including sheep, cattle, camels, and goats slaughtered in Yazd abattoir from September 2018 to January 2020. The demographic data was collected as well as cyst location, fertility, and viability. Out of 657 samples, 164 samples were genotyped. Then, phylogenetic analysis was performed using MEGAX. Statistical analyses were done using SPSS version 16.0 by chi-square with a significant difference of less than 0.05. Results Out of 164 samples, the G1-G3 complex genotype had the most frequency in samples, with 135 cases recognized. The G6/G7 was observed in 19 isolates and G5 was reported in nine samples. One sample was detected as Taenia hydatigena. Conclusions This study showed that G1-G3 and G6/G7 genotypes were presented in all animals, but G5 was reported only in cattle, goats, and camels. It is the first molecular identification of cystic echinococcosis in Central Iran. Hence, reporting G5 in livestock in this area should be considered due to transmission to humans.


Results
The hydatid cysts included in this study were obtained from livestock, including sheep, camels, goats, and cattle. The cyst locations were in both animal liver and lung with the rate of 38.1% in sheep with both lung and liver infection; 38.9% of camels had both lung and liver infection, 25% of cattle had both lung and liver infection, and 50% of goats were infected in both lung and liver. One sample was detected as Taenia hydatigena. The identified E. granulosus s.l. genotypes were G1-G3, G6/G7, and G5. One crucial piece of data shown in this study was that more than one genotype was identified in one animal species infected with hydatid cysts. One goat had two cysts in the lung with two different genotypes of G1-G3 and G6/G7. In cattle, two had cysts in their lungs with two different genotypes of G1-G3 and G5 and one had co-infection of two genotypes of G1-G3 and G6/G7 in the lung. In camels, four animals had co-infection of G1-G3 and G6/G6 genotypes, two had co-infection of G1-G3 and G5 genotypes, and one had three genotypes of G1-G3, G6/G7, and G5. In sheep, co-infection has not been recorded.

Prevalence of infection per cyst location
From 657 hydatid cysts obtained from 97 animals, the distribution of cysts in the lung and liver was almost the same in sheep, goats, and cattle. However, in camels, the lung had more cysts than the liver. Out of 657 cysts, 46 (Fig. 4).
The descriptive analysis showed that 75.3% of the infected animals were female. The chi-square analysis indicated that infection rate and sex had a significant relationship (p = 0.00001). The infection rate at the age of 1 to 5 was 70.1%, and the remaining was regarding the animals more than 5 years old. The statistical analysis showed significant differences among the age

Molecular detection
Out of 657 hydatid cysts, 164 isolates were randomly selected and assessed for molecular genotyping using multiplex PCR (Fig. 5). Finally, out of 164 isolates, 59 were randomly selected for sequencing, and they were amplified by PCR using the specific primer pair of the  gene target of cytochrome c oxidase subunit 1 (cox 1) resulted in the size of the amplicon of 450 bp in length (Fig. 6). The sequences were submitted to NCBI, Gen-Bank (  (3/19) in cattle, and 10.5% (2/19) in goats; and finally, 5.5% (9/163) had G5 genotype, including 44.4% (4/9) in camels, 44.4% (4/9) in cattle, and 11.2% (1/9) in goats. Sequence analysis showed that among E. granulosus s.s. identified in this study, two isolates were identified only as G3 that were in sheep lung. No G5 genotype was found in sheep.
Out of 135 hydatid cysts with G1-G3 genotypes, 54.1% (73/135) samples were in the lung and 45.9% (62/135) were in the liver. The G6/G7 genotype cysts were in the lung and liver, with the rate of 78.9% (15/19) and 21.1% (4/19), respectively. The cysts with the genotype of G5 were in the lung and liver with the rate of 88.9% (8/9) and 11.1% (1/9), respectively. The distribution of the different genotypes was variously based on the location of each animal (Table 2); however, the cyst location in each animal had no significant relationship with genotypes.

Phylogeny analysis
In the Maximum Likelihood (ML) consensus tree obtained, all the specimens here analyzed clustered together with G1-G3 reference strains, demonstrating the circulation of the E. granulosus s.s. in the area (Fig. 7).

Discussion
In this study, 657 hydatid cyst samples were isolated from 63 sheep, 18 camels, 12 cattle, and 4 goats. In 97 infected animals, 73 (75.3%) were females, even though the prevalence of infection was different in animal species (p = 0.00001). A higher prevalence in females was also described in several studies [16][17][18][19][20][21][22][23]. Female animals are at higher risk of CE infection because females are kept longer for milk production and reproductive functions [18]. In addition, females are raised near houses for milking, making them more exposed to infected dogs [19]. The relationship between age and the infected animals was significant in this study (p = 0.00001). The infection rate was high in animals aged between 1 and 5 years, except for camels where high infection rates were seen in animals older than 5 years. The increasing prevalence rate with age is in agreement with other studies carried out in several countries [9,[17][18][19][24][25][26][27][28][29][30]. The causes of infection in older animals are longer duration of exposure and more time for cysts to grow in size for better diagnosis [18]. The cyst develops in camels after the age of 3 [31]. Therefore, we showed that the infection rate is related to the sex and age of animals. All cysts from infected animals were isolated from the lung and liver. We presented that the cyst location had a significant relationship with the infected animal species. Some other results from different studies also show a vast distribution of the cyst in animal organs. Azami et al. [16] reported that the highest and lowest cysts were in sheep lungs (27.1%) and goat liver (11.6%), respectively. In Pakistan, Ehsan et al. [18] demonstrated cysts in cattle, with 8.65% in the liver and 4.80% in the lung. Haleem et al. [19] reported that in Pakistan, the prevalence of hydatid cysts in the liver and lung was 63.49 and 23.80%, respectively, including 69 cattle livers, 9 cattle lungs, 12 sheep livers, 14 sheep lungs, 3 goat livers, and 1 goat lung [19]. In another study in Pakistan, hydatid cysts were distributed in the liver and lung of various animals, including sheep with 67.81% in liver and 32.19% in lung, goats with 66.18% in liver and 32.60 in the lung, cattle with 84.51% in liver and 15.48% in lung, and camels with 83.33% in liver and 16.66% in the lung [32]. In the other study in Pakistan, the infection rates were 46.74% in sheep liver, 23.28% in goat liver, 17.37% in sheep lung, and 13.68% in goat lung [33]. Singh et al. [34] reported liver and lung infection rates with 49.66 and 36.179%, respectively, in India's cattle, buffaloes, sheep, and goats. Elmajdoub and Rahman [35] in Libya demonstrated that among 372 samples in different organs, including 135 sheep livers, 105 sheep lungs, 28 camel livers, 64 camel lungs, 6 cattle livers, and 6 lung cattle. In a study carried out in north Ethiopia, hydatid cysts in cattle were 13% in the lung and 8.1% in the liver [36]. The organs' involvement is because passing the oncosphere is carried out via portal vein flow into the liver and other organs [37].
A high fertility rate was observed in cysts isolated from sheep, followed by goats and camels. No fertility was reported in the samples from cattle. The fertility rate of hydatid cysts was higher in the liver of sheep, whereas in camels and goat, fertile cysts were mostly found in lungs. Some studies have reported more fertility in the liver [16,20,[38][39][40], and some studies reported more fertility in the lung [41][42][43][44]. In this study, it was found that none of the cysts from the cattle were fertile. This result is in agreement with some other studies [30,45,46]. No fertility in the hydatid cysts of the cattle reveals that this intermediate host would not be considered suitable, and therefore, cattle are a dead-end host [47]. Hence, understanding the fertility and viability of the hydatid cysts in the intermediate host is critical because livestock play as the main source of infection transmission of the final host by consuming fertile cysts. The fertility of the hydatid cyst is different in various geographical situations, animal species, locations, size, and genotype of the cyst [48]. There are many reports about the infertility rate of the hydatid cysts in various genotypes and animal species in different regions [36,49], indicating that fertility depends on the genotype. The fertility rate may be different in various hosts due to the immunological response in the hosts [50]. In our study, high rate of cyst viability was observed in sheep, goats, and camel, in agreement with other studies [16,19,51]. This study was the first molecular detection and identification of E. granulosus s.l. in slaughtered livestock in a desert area in Central Iran. However, more molecular epidemiological studies have been done on hydatid cysts in intermediate hosts in some other areas of Iran [8]. The only limitation in this study is that the molecular characterization was not performed on all hydatid cysts. Genotyping analysis in this study showed that E. granulosus s.l. G1-G3 genotypes were the most frequent in this area in all animals.
Out  [68] reported the G6 genotype in goats in Iran. Kesik et al. [69] reported one sample with the G6 genotype obtained from a camel in Turkey. There are also some reports of G6 genotype in goat in Argentina [70,71]. The main intermediate host for the G6 genotype is the camel. However, it seems that camels living with other livestock may expose to other genotypes through interaction with dogs as definitive hosts living near them. There are some reports of G6 in humans worldwide. The first report of E. Canadensis (G6/G7) in humans found in Pakistan shows the importance of this genotype in clinical aspects [72], even though it was thought that the G6 genotype might be less infective to humans [73]. However, it is considered as the second most important causative agent of CE after E. granulosus s.s [74]. In South America, Africa, and Asia; E. granulosus s.l. G6 genotype infects humans [73].
Simsek and Kaplan [75] also reported two cases of human infection with the G6 genotype in Turkey.  E. ortleppi (G5) was not reported from sheep, but it was identified from nine cysts, one belonging to goats (11.2%), four to camels (44.4%), and four to cattle (44.4%). This result is consistent with other studies done in different regions of Iran [52][53][54][55][56][57][58]. As mentioned above, E. ortleppi (G5) was reported in 5.5% (9/163) of E. granulosus s.l. cysts from goats, camels, and cattle that is less than the study by Nematdoost et al. [62] reported G5 in 5/45 (11.1%) of cattle. Some other studies reported G5 genotype in buffaloes [76], goats [77], and camels [77][78][79]. Moreover, some studies reported G5 genotype in sheep [77,80] that is opposite to our results. In this study, 75% (3/4) of hydatid cysts found in camels with G5 genotype were fertile, whereas G5 in goats and cattle were infertile. The result concerning the G5 genotype showing no fertility in cattle agrees with the study conducted in Pakistan [81]. However, Monteiro et al. [82] reported the frequency of 43.4% G5 genotype for fertile cysts in the lung of cattle in Brazil.

Conclusions
To the best of our knowledge, the molecular identification of hydatid cysts was performed for the first time in the livestock slaughtered in Central Iran. The genotypes of G1-G3 and G6/G7 exist in all livestock being studied, but G5 was reported only in cattle, goats, and camels. Therefore, the dominant genotypes in Central Iran are G1-G3 and then G6/G7, which should be considered a high-priority public health concern.

Methods
In this study, 97 slaughtered animals with echinococcosis from Yazd abattoir were included to assess the variables of sex, age, cyst location, fertility, viability of  The sex and age of each animal were recorded by a veterinarian in the slaughterhouse. The eruption of permanent incisor teeth was the main criteria used to determine the age of the animals [83].

Viability and fertility
After sterilizing the cyst surface with 70% alcohol, the cyst fluid was aspirated and centrifuged at 500 xg for 60 s. For fertility assessment, the pellet was analyzed for protoscoleces present using a light microscope. Cysts without any protoscoleces were detected as sterile.
The viability of the protoscoleces was evaluated using 0.1% eosin staining. The nonviable protoscoleces were stained in red.

DNA extraction
Protoscoleces (if presented) and germ layers were used for genotype analysis. The protoscoleces were washed with phosphate-buffered solution (PBS) in triplicate. According to the manufacturer's instructions, the genomic DNA (gDNA) was extracted using a Tissue DNA Extraction Kit (GeneAll, South Korea). The quantity of the extracted DNA samples was analyzed using a NanoDrop device (Thermo Fisher Scientific, Massachusetts, USA) and stored at − 20 °C until the next analysis.

Molecular detection
Characteristics of primers used for multiplex PCR are shown in Table 3

Sequencing
To verify the multiplex PCR resulting from genotyping, 59 samples were selected to sequence the gene target of cox 1 randomly from the cysts obtained from all various species of animals. The PCR reaction was conducted using a thermal cycler (ABI, USA) in a 20 μl final volume containing 50-100 ng gDNA, 0.2 mM dNTPs (Ampliqon, Denmark), 1.5 mM MgCl 2 (Ampliqon, Denmark), 10 pmol of each primer (ordered from Pishgam Company), 1.5 U Taq DNA polymerase, and ddH 2 O up to the final volume. Amplification was done using the specific primer pair for cox 1 by JB3: 5′-TTT TTT GGG CAT CCT GAG GTT TAT -3′ and JB4.5: 5′-TAA AGA AAG AAC ATA ATG AAA ATG -3′ [84] resulted in an amplicon fragment of 450 bp in length. The temperature conditions were an initial denaturation at 94 °C for 5 min; then 35 cycles of denaturation at 94 °C for 45 s, annealing at 57 °C for 45 s, and elongation at 72 °C for 45 s. The final extension was done at 72 °C for 5 min. PCR products were detected in 1% agarose gel electrophoresis (Akhtarian, Tehran, Iran). The PCR product was excised from agarose gel, and sent to the Company (Pishgam Company, Tehran, Iran) for purification and sequencing. The sequencing results were analyzed using BLAST.

Phylogenetic analysis
Sequence data of E. granulosus s.l. isolates were complete alignment using the T-COFFEE software [85]. Also haplotypes defined by combination of single nucleotide polymorphisms were assigned using DnaSP V.6. Phylogenetic analysis were inferred from DNA sequences using Maximum Likelihood (ML) estimates with MEGAX based on Tamura-Nei model with 1000 bootstrap replicates. All positions containing gaps and missing data were eliminated.

Statistical analysis
The obtained data were analyzed using SPSS version 16.0. In the first step, the data were entered into the software. Then, descriptive data analysis was performed, including frequency and prevalence calculation. The relationship between infection in animals with sex, age, cyst location, and fertility, and viability of infected animal species were analyzed using the chi-square test.
With confidence level of 95%, P-value < 0.05 was considered as significant.